It is now widely accepted, given the current weight of experimental evidence, that reactive oxygen species (ROS) contribute to cell and tissue dysfunction and damage caused by glucolipotoxicity in diabetes. The source of ROS in the insulin secreting pancreatic β-cells and in the cells which are targets for insulin action has been considered to be the mitochondrial electron transport chain. While this source is undoubtably important, we provide additional information and evidence for NADPH oxidase-dependent generation of ROS both in pancreatic β-cells and in insulin sensitive cells. While mitochondrial ROS generation may be important for regulation of mitochondrial uncoupling protein (UCP) activity and thus disruption of cellular energy metabolism, the NADPH oxidase associated ROS may alter parameters of signal transduction, insulin secretion, insulin action and cell proliferation or cell death. Thus NADPH oxidase may be a useful target for intervention strategies based on reversing the negative impact of glucolipotoxicity in diabetes.
Aims/hypothesis Acute or chronic exposure of beta cells to glucose, palmitic acid or pro-inflammatory cytokines will result in increased production of the p47 phox component of the NADPH oxidase and subsequent production of reactive oxygen species (ROS). Methods Rat pancreatic islets or clonal rat BRIN BD11 beta cells were incubated in the presence of glucose, palmitic acid or pro-inflammatory cytokines for periods between 1 and 24 h. p47 phox production was determined by western blotting. ROS production was determined by spectrophotometric nitroblue tetrazolium or fluorescencebased hydroethidine assays.
The presence of a phagocyte-like NAD(P)H oxidase in pancreatic -cells was investigated. Three NAD(P)H oxidase components were found in pancreatic islets by RT-PCR: gp91 PHOX , p22 PHOX , and p47 PHOX . The components p67PHOX and p47 PHOX were also demonstrated by Western blotting. Through immunohistochemistry, p47PHOX was mainly found in the central area of the islet, confirming the expression of this component by insulin-producing cells. Activation of NAD(P)H oxidase complex in the -cells was also examined by immunohistochemistry. The pancreatic islets presented slower kinetics of superoxide production than HIT-T15 cells, neutrophils, and macrophages, but they reached 66% that of the neutrophil nitroblue tetrazolium (NBT) reduction after 2 h of incubation. Glucose (5.6 mmol/l) increased NBT reduction by 75% when compared with control. The involvement of protein kinase C (PKC) in the stimulatory effect of glucose was confirmed by incubation of islets with phorbol myristate acetate (a PKC activator) and bysindoylmaleimide (GF109203X) (a PKC-specific inhibitor). Diphenylene iodonium [an NAD(P)H oxidase inhibitor] abolished the increase of NBT reduction induced by glucose, confirming the NAD(P)H oxidase activity in pancreatic islets. Because reactive oxygen species are involved in intracellular signaling, the phagocyte-like NAD(P)H oxidase activation by glucose may play an important role for -cell functioning.
It is now widely accepted that reactive oxygen species (ROS) contribute to cell and tissue dysfunction and damage in diabetes. The source of ROS in the insulin secreting pancreatic beta cells has traditionally been considered to be the mitochondrial electron transport chain. While this source is undoubtedly important, we fully describe in this article recent information and evidence of NADPH oxidase-dependent generation of ROS in pancreatic beta cells and identify the various isoforms that contribute to O 2•− and H 2 O 2 production in various conditions. While glucose-stimulated ROS generation may be important for acute regulation of insulin secretion, at higher levels ROS may disrupt mitochondrial energy metabolism. However, ROS may alter other cellular processes such as signal transduction, ion fluxes and/or cell proliferation/death. The various beta cell isoforms of NADPH oxidase (described in this review) may, via differences in the kinetics and species of ROS generated, positively and negatively regulate insulin secretion and cell survival. Regulation of insulin secretionThe combination of glucose transport and phosphorylation at the glucokinase step determines a major part of metabolic flux through glycolysis in the beta cell. Glucose may be further metabolised by pyruvate dehydrogenase and pyruvate carboxylase, resulting in enhanced tricarboxylic acid cycle activity and subsequent ATP generation [1]. This results in closure of the ATP-sensitive K + channels, decreasing the hyperpolarising outward K + flux, depolarising the plasma membrane and opening voltage-gated Ca 2+ channels to increase the influx of extracellular Ca 2+ , which is a primary driver of the mechanism of glucose-stimulated P. Newsholme (*)
We previously described the presence of nicotinamide adenine dinucleotide phosphate reduced form [NAD(P)H]oxidase components in pancreatic beta-cells and its activation by glucose, palmitic acid, and proinflammatory cytokines. In the present study, the importance of the NAD(P)H oxidase complex for pancreatic beta-cell function was examined. Rat pancreatic islets were incubated in the presence of glucose plus diphenyleneiodonium, a NAD(P)H oxidase inhibitor, for 1 h or with the antisense oligonucleotide for p47(PHOX) during 24 h. Reactive oxygen species (ROS) production was determined by a fluorescence assay using 2,7-dichlorodihydrofluorescein diacetate. Insulin secretion, intracellular calcium responses, [U-(14)C]glucose oxidation, and expression of glucose transporter-2, glucokinase and insulin genes were examined. Antisense oligonucleotide reduced p47(PHOX) expression [an important NAD(P)H oxidase cytosolic subunit] and similarly to diphenyleneiodonium also blunted the enzyme activity as indicated by reduction of ROS production. Suppression of NAD(P)H oxidase activity had an inhibitory effect on intracellular calcium responses to glucose and glucose-stimulated insulin secretion by isolated islets. NAD(P)H oxidase inhibition also reduced glucose oxidation and gene expression of glucose transporter-2 and glucokinase. These findings indicate that NAD(P)H oxidase activation plays an important role for ROS production by pancreatic beta-cells during glucose-stimulated insulin secretion. The importance of this enzyme complex for the beta-cell metabolism and the machinery involved in insulin secretion were also shown.
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